Mass spectrometers are each a device of ionizing a sample molecule, separating its resultant ions through an electric field or a magnetic field in accordance with their mass-to-charge ratios, and then, measuring the quantity of separated specific ions as a current by a detector. That is: a sample is separated into individual components respectively through a separation column of a liquid chromatograph and then introduced into an ionization unit; in an ionization unit of amass spectrometer, molecules of the sample is ionized, and the ionized ions are introduced into the mass spectrometer; the introduced ions are trapped in an electric field made of its ion trap unit; the trapped ions are ejected selectively from the ion trap unit in accordance with their mass-to-charge ratios or ejected together from the ion trap unit; ant then the ejected ions are detected in its detection unit. A graph in which the mass-to-charge ratio of each of the ions detected in this unit is taken as a transverse axis and its signal intensity is taken as a vertical axis is called an MS spectrum (mass spectrum).
Each mass spectrum has data as to a specific mass-to-charge ratio of its corresponding ion in a sample molecule and signal strength thereof, and structural information on the sample component can be gained from such data. When carrying out an analysis of peptides, reading out information as to the peptides contained in a sample from peak data represented in an MS spectrum, and then carrying out the analysis by using a computer and an exclusive data base or some other. In order to raise precision of the analysis, it is necessary to obtain a larger number of structural information from the MS spectrum.
However, in a case that components in a sample have complex structures, when, in spite of the fact that the components are different from each other in structure, they have an equal mass-to-charge-ratio, the difference therebetween may not be sufficiently discerned only by information obtained from their MS spectra. In such a case, executed is measurement according to tandem mass spectrometry (hereinafter referred to as MS/MS analysis) (see, for example, Patent Document 1).
In MS/MS analysis, the method is comprised of: trapping ions into an ion trap; excluding non-target ions therein by applying energy such as high-frequency voltage; subjecting selectively left ions to dissociation by making them collide with neutral molecules such as rare gas molecules thereby to break bonds of the molecular ion; and measuring resultant dissociated ions (called as fragment ions). This dissociation, which is generated by the collision with the neutral molecules, is called as collision induced dissociation (abbreviated to CID), thereby obtained is an MS/MS spectrum. An analyzing operation of repeating sequential operations for MS/MS analysis is called as MS2, MS3 or the like in accordance with the number of times of the repeating. From peculiar fragment ions generated by this operation, a larger number of structural information can be obtained about the sample.
When ions to be dissociated in execution of MS/MS analysis are beforehand known, an analyzer can set the ions as conditions to be used in the measurement. However, in a protein analysis or some other analysis, it is unclear what ions are contained in a sample in many cases. In such a case, the analyzer initially subjects the sample to an MS analysis, subsequently determines, from an MS spectrum gained therein, ions to be dissociated under certain conditions, and then subjects the determined ion to an MS/MS analysis. By repeating this operation, structural information can be gained about many ions to be measured.
In a liquid chromatograph, a sample is separated into individual components through its separation column in accordance with the degree of affinity thereof for the separation column, and thereby, the components are successively eluted out from the separation column. These components are each introduced into a mass spectrometer to gain an MS spectrum. A graph where signal intensities at a specific mass-to-charge ratio are lined with a time series is referred to as a mass chromatogram.
When examining what quantity of a detected component is contained in a sample, a measurement is carried out beforehand about a reference sample in which a concentration of this kind of component is already known, and then a calibration curve is prepared on a basis of the area or height of a peak of the component on a mass chromatogram obtained in the measurement to carry out a quantitative calculation of the component. In recent years, mass spectrometers have been required to have a function of using peaks of fragments subjected to MS/MS analysis to carry out such a quantitative determination.
In order to carry out an MS analysis for gaining an MS spectrum, the following operation is made in an ion trap. Ions introduced into amass spectrometer are further introduced into an ion trap unit, and then trapped by an electric field made in the ion trap unit (accumulation). This electric field is changed to eject the ions from the ion trap unit (ejection), and then the ions are detected in a detector. In order to conduct an MS/MS analysis, the following operation is carried out in the ion trap unit.
Ions introduced in the mass spectrometer are introduced into an ion trap unit, and trapped by an electric field generated in the ion trap unit (accumulation). Non-target ions are excluded from the ions trapped in the ion trap unit (isolation), and then energy such as high-frequency voltage, corresponding to the selectively left ion therein, is applied to cause impact induced dissociation (CID). Subsequently, the electric field in the ion trap unit is changed to eject the ion from the ion trap unit, and then the ion is detected in the detector (ejection).
In MSn analysis in which n thereof represents the number of times of MS, the isolation and the CID are performed plural times between the accumulation and the ejection, thereby, target ions can be detected.
In MS/MS analysis, there is time required for isolations and CIDs, so that a period necessary for gaining an MS spectrum becomes longer than in MS analysis. In the meantime, the width of any peak in a chromatogram is fixed depending on the performance of the separation column. Thus, the number of MS spectra that can be gained in this period is limited.
Fragment ions of an MS spectrum that are gained as a result of an MS/MS analysis, each has a smaller signal intensity than strength of originally selected ion. When structural information is gained from the MS spectrum, a given threshold for separating signals from noises is set in order to avoid effects of the noises and others. Thereby, the structural information is gained from any peak equal or larger (in value) than the threshold. In such a way, an MS spectrum having a sufficient strength can be gained.
In order to identify a fragment pattern precisely from an MS spectrum, it is desired to subject the concerned component to MS/MS analysis at plural times to render its ion strength information sufficient information. Therefore, it is required to carry out MS/MS analysis at plural times during a component per one elutes out from the separation column. When two or more components elute out in the same time band from the column, it is required to subject these components to respective MS/MS analyses while the components elute out therefrom.
As described above, MS/MS analysis is required to be carried out in a short period. However, in the MS/MS analysis, operations of isolations and CIDs are since necessary, more time is required to gain a MS spectrum of a component per one therein than in MS analysis. Furthermore, recent liquid chromatographs have been promoted in operation-speed to improve performance of their separation column for separating a sample into individual components, and reduce a consumed volume of a solvent of each chromatograph.
In such a high-speed liquid chromatograph, a period when each component elutes out has become shorter, so that a further restriction is imposed onto the number of times of MS/MS analysis that can be carried out by a mass spectrometer in the period when the component per one elutes out. Accordingly, the mass spectrometer has been desired to make shorter a period required for gaining each MS/MS spectrum and gain MS spectra as many as possible.
When dissociation is performed to make an MS/MS analysis in an analysis of a protein or some other, it is unclear what ions are contained in a sample. Therefore, when performing the dissociation, it is performed by the following method of: first of all, making an MS analysis initially; determining, from an MS spectrum gained therein, an ion to be dissociated under some condition; and subjecting the determined ion to an MS/MS analysis. By repeating this method, structural information can be gained about many ions to be measured.
In the method, the MS analysis and the MS/MS analysis are different from each other in period necessary for analysis. For this reason, these analyses do not become identical with each other in sampling-period of the chromatogram, so that the same components having the same concentration are varied in peak area. The matter that the same components having the same concentration are different from each other in peak area results in a problem when a quantitative calculation is made.